Mammalian pro-apoptotic Bok genes and their uses

ABSTRACT

Nucleic acid compositions encoding a pro-apoptotic protein, Bok (Bcl-2-related ovarian killer) are identified. Bok has conserved Bcl-2 homology domains 1, 2 and 3 and a C-terminal transmembrane region present in other Bcl-2 related proteins, but lacks the BH4 domain found only in anti-apoptotic Bcl-2 proteins. Over-expression of Bok induces apoptosis. Cell killing induced by Bok is suppressed by co-expression with selective anti-apoptotic Bcl-2 proteins. Bok is highly expressed in the ovary, testis and uterus, particularly in granulosa cells, the cell type that undergoes apoptosis during follicle atresia. Identification of Bok as a new pro-apoptotic protein with wide tissue distribution and hetero-dimerization properties facilitates elucidation of apoptosis mechanisms in reproductive and other tissues, and provides a means for manipulating apoptosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.09/186,250, filed Nov. 4, 1998, now U.S. Pat. No. 6,043,055 which claimspriority to U.S. provisional patent application No. 60/064,943, filedNov. 7, 1997.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant no.HD-31566, awarded by the National Institutes of Health. The Governmentmay have certain rights in this invention.

BACKGROUND

Apoptosis or programmed cell death is important during embryonicdevelopment, metamorphosis, tissue renewal, hormone-induced tissueatrophy and many pathological conditions. In multi-cellular organisms,apoptosis ensures the elimination of superfluous cells including thosethat are generated in excess, have already completed their specificfunctions or are harmful to the whole organism. In reproductive tissuesthat are characterized by cyclic functional changes, massive cell deathoccurs under the control of hormonal signals. A growing body of evidencesuggests that the intracellular “death program” activated duringapoptosis is similar in different cell types and conserved duringevolution.

Apoptosis involves two essential steps. The Bcl-2 family of proteinsthat consists of different anti- and pro-apoptotic members is importantin the “decision” step of apoptosis. In contrast, the “execution” phaseof apoptosis is mediated by the activation of caspases, cysteineproteases homologous to the C. elegans protease ced-3, that induce celldeath via the proteolytic cleavage of substrates vital for cellularhomeostasis. Bcl-2-related proteins act upstream from caspases in thecell death pathway and recent studies demonstrated that another C.elegans gene, ced-4, or its mammalian homolog Apaf-1 can bridge betweenBcl-2/ced-9 family members and caspases.

The proto-oncogene Bcl-2 was originally isolated at the breakpoint ofthe t(14, 18) chromosomal translocation associated with follicularB-cell lymphoma. Over-expression of Bcl-2 suppresses apoptosis inducedby a variety of agents both in vitro and in vivo. Subsequent studiesidentified a family of Bcl-2-related proteins possessing severalconserved BH (Bcl-2 homology) domains important for homo- orhetero-dimerization between family members. In addition, a C-terminaltransmembrane region for membrane anchoring is also conserved in mostmembers. Based on their differential roles in regulating apoptosis, theBcl-2-related proteins can be separated into anti-apoptotic (Bcl-2,Bcl-xL, Mcl-1, Bcl-w and Bfl-1/A1) and pro-apoptotic members (Bax, BAD,Bak, Bik, Hrk and BID). Through hetero-dimerization, the balance betweenpro- and anti-apoptotic proteins presumably determines cell fate. Theanti-apoptotic effect of Bcl-2 is not universal, however, because Bcl-2over-expression is not effective in blocking Fas-mediated apoptosis andthe apoptosis of auto-reactive thymocytes during negative selection.Recent identification of multiple Bcl-2-related proteins suggests thatselective Bcl-2 members may act in a tissue- and dimerization-specificmanner.

References

Bcl related genes are discussed in Yin et al. (1994) Nature 369:321-323;Chittenden et al. (1995) EMBO J. 14:5589-5596; and White (1996) GenesDev. 10:1-15.

Sequences of exemplary bcl-related genes may be accessed in Genbank. Thehuman hrk gene has the accession no. U76376 and is described in Inoharaet al. (1997) EMBO J. 16:1686-1694. The human bcl-w gene has theaccession no. U59747 and is described in Gibson et al. (1996) Oncogene13:665-675. Human A1 gene has the accession no. U29680, and is describedin Karsan et al. (1996) Blood 87:3089-3096. The human Bak gene has theaccession no. U23765, and is described in Chittenden et al. (1995)Nature 374:733-736. The human Bak-2 gene has the accession no. U16812,and is described in Kiefer et al. (1995) Nature 374:736-739. The humanBik gene has the accession no. U34584, and is described in Boyd et al.(1995) Oncogene 11:1921-1928. The human Bfl-1 gene has the accession no.U27467, and is described in Choi et al. (1995) Oncogene 11:1693-1698.The human bcl-2 gene has the accession no. M13995, and is described inTsujimoto and Croce (1986) P.N.A.S. 83:5214-5218. The human Bax geneshave the accession nos. L22475, L22474 and L22473, and are described inOltvai et al. (1993) Cell 74:609-619. The EBV BHRF1 gene has theaccession no. A22899, and is described in WO 9311267. The human mcl-1gene is described in Kozopas et al. (1993) P.N.A.S. 90:3516-3520, andOMIM 159552.

The EST fragment, Genbank accession no. AA103989, contains partialsequence of the 5′ end of the mouse Bok gene.

SUMMARY OF THE INVENTION

Isolated nucleotide compositions and sequences are provided for Bokgenes. The provided nucleic acids include splice variants encoding longforms of the protein, as well as short forms having a truncation thatdeletes all or a part of the BH3 domain. The short form of Bok and otherrelated pro-apoptotic proteins may be naturally occurring or synthetic.These short forms induce cell killing without heterodimerization withantiapoptotic proteins.

The Bok nucleic acid compositions find use in identifying homologous orrelated genes; in producing compositions that modulate the expression orfunction of its encoded protein; for gene therapy; mapping functionalregions of the protein; and in studying associated physiologicalpathways. In addition, modulation of the gene activity in vivo is usedfor prophylactic and therapeutic purposes, such as treatment of cancerand other proliferative disorders, identification of cell type based onexpression, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing quantitative analysis of cell killing by Bokand the inhibitory effects of P35. The number of β-gal-expressing cells(mean+/−SEM, n=3) was determined at 36 h after transfection. Data fromcells transfected with three independent clones (1, 2 and 3) encodingBok are presented. CHO cells were transfected with a total of 2.1 μgplasmid DNA including 2.0 μg of pcDNA3 expression constructs and 0.1 μgof the pCMV-β-gal reporter. In cells transfected with two differentpcDNA3 expression plasmids, 1.0 μg each was used. Similar results wereobtained in three separate experiments.

FIG. 2 is a graph showing suppression of Bok-induced apoptosis byselective anti-apoptotic Bcl-2 members in CHO cells. Cell killing by Bokand the antagonistic effects of Mcl-1 and BHRF1 were analyzed. Celltransfection and estimation of apoptosis were as described for FIG. 1.Co-expression of Bcl-2 was ineffective in suppressing Bok-inducedapoptosis.

FIG. 3 is a schematic representation of wild type Bax and Bak togetherwith Bax-S and Bak-S constructs with BH3-BH1 deletions similar to thatfound in Bok-S. The BH domains are boxed and the junctional sequencesderived from the fusion of BH3 and BH1 domains (BH3/1) in the mutantsare also shown. Numbering of amino acid in the BH1, BH3 and BH3/1domains are indicated at the bottom of amino acid residues.

DETAILED DESCRIPTION OF THE INVENTION

Nucleic acid compositions encoding Bok, a pro-apoptotic member of thebcl-2 protein family, are provided. Included are splice variantsencoding long forms of the protein, as well as short forms having atruncation that deletes all or a part of the BH3 domain. Also providedare truncated forms of pro-apoptotic proteins related to Bok, e.g. Bax,Bak, etc. These short forms may be naturally occurring or synthetic. Thelong forms associate with anti-apoptotic proteins to form heterodimers,while the short forms induce cell killing without suchheterodimerization.

As used herein, the term “Bok” is intended to generically refer to thepolypeptide or nucleic acids as set forth in the Seqlist attachedherewith, homologs thereof, and sequences having substantial similarityand function. Bok occurs naturally in a long form (herein Bok-L), asexemplified by the amino acid sequences provided in SEQ ID NO:2 and SEQID NO:6, which are rat and human, respectively. A short form (hereinBok-S) also occurs naturally, as exemplified by SEQ ID NO:4 and SEQ ID.NO:8, in which there is a deletion, leading to the fusion of theN-terminal half of the BH3 domain to the C-terminal half of the BH1domain (herein, BOK-BH3^(inactive)).

The term “BH3_(inactive)”, or “BH3^(i)” is intended to generically referto naturally occurring splice variants and synthetic variants of Bok orpro-apoptotic Bok-related proteins, e.g. Bax, Bak, etc., in whichdeletions or amino acid substitutions made in the BH3 domainsubstantially inactivate or abrogate the heterodimerization activity ofthe protein. These variants may also be referred to as “channel only”proteins, because they retain the ability to form channels in themitochondria that promote apoptosis.

The BH3^(i) variants will usually have at least less than about 50% ofthe anti-apoptotic protein binding activity of the parent “long” form,more usually less than about 75% of the anti-apoptotic protein bindingactivity, and preferably less than about 95% of anti-apoptotic proteinbinding activity. Examples are provided herein of BH3^(i) variants,including but limited to: alanine substitutions at the highly conservedBok glycine 75 residue, truncations of Bax and Bak in the BH3 domain,splice variants of Bok where there is a deletion of the amino acids76-118; and a glycine substitution was made for leucine 71 to leucine 74(BokGGGG: 71 LLRL 74 to 71 GGGG 74).

The BH3 domain has the consensus motif sequence: [SEQ ID NO:11}LRRAGDEFE.RYRR, and generally corresponds to the region of amino acids71-82, in Bok (SEQ ID NO:9 and SEQ ID NO:10). A substitution at theconserved gly75 residue is shown to be sufficient for inactivation.

Modulation of pro-apoptotic gene activity, which may include Bok orother pro-apoptotic BH3^(i) variants, in vivo is used for prophylacticand therapeutic purposes where it is desirable induce cell death inspecific populations. The specificity of Bok for reproductive tissues isparticularly useful in this respect. Diseases where there ishyperproliferation of reproductive tissue, e.g. uterine, testicular andovarian carcinomas, endometriosis, squamous and glandular epithelialcarcinomas of the cervix, etc. are reduced in cell number byupregulating Bok expression to cause apoptosis in susceptible cells.Expression can be regulated by introduction of exogenous Bok genes, orby inducing expression of the native gene. Introduction of exogenous Bokgene and its channel domain only variants into tumor cells followingdirect injection or using tumor-specific carriers can serve as effectivetherapies.

The isolated Bok genes are useful for in vitro, i.e. cell culture orcell-free assays, investigation of apoptosis pathways, identification ofcell type based on expression, and the like. The protein is useful as animmunogen for producing specific antibodies, in screening forbiologically active agents that act to regulate Bok gene expression, orthat directly mimic, agonize or antagonize Bok protein function.

Characterization of Bok

Bok is expressed mainly in mammalian reproductive tissues, includingovary, testis and uterus. Bok is also expressed in diverse othertissues, albeit at lower levels. It forms a heterodimer with specificanti-apoptotic Bcl-2 proteins, including mcl-1, BHRF1 and Bfl-1. Theprotein-protein interaction is mediated by the conserved BH1, 2 and 3domain regions of Bok, particularly by the BH3 domain. Over-expressionof Bok induces apoptosis in certain cells, particularly reproductivecells. The rat cDNA sequence is provided as SEQ ID NO:1, the encodedpolypeptide product as SEQ ID NO:2. The gene encodes a 213 amino acidpolypeptide. The rat short form is provided as SEQ ID NO:3, the encodedpolypeptide as SEQ ID NO:4. The nucleotide sequences of the human longand short forms are provided as SEQ ID NO:5 and 7; the encodedpolypeptides as SEQ ID NO:6 and 8.

Many members of the bcl-2 gene family have been identified andcharacterized, as previously indicated. Other proteins in the pathwayhave also been identified, including caspases, and Apaf-1. Theavailability of isolated genes and gene products in this pathway allowsthe in vitro reconstruction of the pathway and its regulation, usingnative or genetically altered molecules, or a combination thereof. Alsoof interest is the use of the genomic region 5′ to Bok or related genes,particularly those members that are hormonally regulated, in order toinvestigate the role of particular transcription factors in regulatingexpression.

Identification of Bok Sequences

Homologs of Bok are identified by any of a number of methods. A fragmentof the provided cDNA may be used as a hybridization probe against a cDNAlibrary from the target organism of interest, where low stringencyconditions are used. The probe may be a large fragment, or one or moreshort degenerate primers.

Nucleic acids having sequence similarity are detected by hybridizationunder low stringency conditions, for example, at 50° C. and 10XSSC (0.9M NaCl/0.09 M sodium citrate) and remain bound when subjected to washingat 55° C. in 1XSSC. Sequence identity may be determined by hybridizationunder stringent conditions, for example, at 50° C. or higher and 0.1XSSC(9 mM NaCl/0.9 mM sodium citrate). Nucleic acids that are substantiallyidentical to the provided Bok sequences, e.g. allelic variants,genetically altered versions of the gene, etc., bind to the provided Boksequences under stringent hybridization conditions. By using probes,particularly labeled probes of DNA sequences, one can isolate homologousor related genes. The source of homologous genes may be any species,e.g. primate species, particularly human; rodents, such as rats andmice, canines, felines, bovines, ovines, equines, yeast, nematodes, etc.

Between mammalian species, e.g. human and mouse, homologs havesubstantial sequence similarity, i.e. at least 75% sequence identitybetween nucleotide sequences. Sequence similarity is calculated based ona reference sequence, which may be a subset of a larger sequence, suchas a conserved motif, coding region, flanking region, etc. A referencesequence will usually be at least about 18 nt long, more usually atleast about 30 nt long, and may extend to the complete sequence that isbeing compared. Algorithms for sequence analysis are known in the art,such as BLAST, described in Altschul et al. (1990) J Mol Biol215:403-10. The sequences provided herein are essential for recognizingBok related and homologous proteins in database searches.

Bok Nucleic Acid Compositions

Nucleic acids encoding Bok may be cDNA or genomic DNA or a fragmentthereof. The term “Bok gene” shall be intended to mean the open readingframe encoding specific Bok polypeptides, e.g. splice variants; introns;as well as adjacent 5′ and 3′ non-coding nucleotide sequences involvedin the regulation of expression, up to about 20 kb beyond the codingregion, but possibly further in either direction. The gene may beintroduced into an appropriate vector for extrachromosomal maintenanceor for integration into a host genome.

The term “cDNA” as used herein is intended to include all nucleic acidsthat share the arrangement of sequence elements found in native maturemRNA species, where sequence elements are exons and 3′ and 5′ non-codingregions. Normally mRNA species have contiguous exons, with theintervening introns, when present, removed by nuclear RNA splicing, tocreate a continuous open reading frame encoding a Bok protein.

A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It may further include the 3′ and 5′untranslated regions found in the mature mRNA. It may further includespecific transcriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5′ or 3′ end of the transcribedregion. The genomic DNA may be isolated as a fragment of 100 kbp orsmaller; and substantially free of flanking chromosomal sequence. Thegenomic DNA flanking the coding region, either 3′ or 5′, or internalregulatory sequences as sometimes found in introns, contains sequencesrequired for proper tissue and stage specific expression.

The sequence of the 5′ flanking region may be utilized for promoterelements, including enhancer binding sites, that provide fordevelopmental regulation in tissues where Bok is expressed. The tissuespecific expression is useful for determining the pattern of expression,for providing promoters that mimic the native pattern of expression, andfor determination of transcription factors that regulate expression.Naturally occurring polymorphisms in the promoter region are useful fordetermining natural variations in expression, particularly those thatmay be associated with disease.

Alternatively, mutations may be introduced into the promoter region todetermine the effect of altering expression in experimentally definedsystems. Methods for the identification of specific DNA motifs involvedin the binding of transcriptional factors are known in the art, e.g.sequence similarity to known binding motifs, gel retardation studies,etc. For examples, see Blackwell et al. (1995) Mol Med 1: 194-205;Mortlock et al. (1996) Genome Res. 6: 327-33; and Joulin and Richard-Foy(1995) Eur J Biochem 232: 620-626.

The regulatory sequences may be used to identify cis acting sequencesrequired for transcriptional or translational regulation of Bokexpression, especially in different tissues or stages of development,and to identify cis acting sequences and trans acting factors thatregulate or mediate Bok expression. Such transcription or translationalcontrol regions may be operably linked to a Bok gene in order to promoteexpression of wild type or altered Bok or other proteins of interest incultured cells, or in embryonic, fetal or adult tissues, and for genetherapy. Expression of Bok may be regulated through hormonal control.

The nucleic acid compositions of the subject invention may encode all ora part of the subject polypeptides. Double or single stranded fragmentsmay be obtained of the DNA sequence by chemically synthesizingoligonucleotides in accordance with conventional methods, by restrictionenzyme digestion, by PCR amplification, etc. For the most part, DNAfragments will be of at least 15 nt, usually at least 18 nt or 25 nt,and may be at least about 50 nt. Such small DNA fragments are useful asprimers for PCR, hybridization screening probes, etc. Larger DNAfragments, i.e. greater than 100 nt are useful for production of theencoded polypeptide. For use in amplification reactions, such as PCR, apair of primers will be used. The exact composition of the primersequences is not critical to the invention, but for most applicationsthe primers will hybridize to the subject sequence under stringentconditions, as known in the art. It is preferable to choose a pair ofprimers that will generate an amplification product of at least about 50nt, preferably at least about 100 nt. Algorithms for the selection ofprimer sequences are generally known, and are available in commercialsoftware packages. Amplification primers hybridize to complementarystrands of DNA, and will prime towards each other.

The Bok genes are isolated and obtained in substantial purity, generallyas other than an intact chromosome. Usually, the DNA will be obtainedsubstantially free of other nucleic acid sequences that do not include aBok sequence or fragment thereof, generally being at least about 50%,usually at least about 90% pure and are typically “recombinant”, i.e.flanked by one or more nucleotides with which it is not normallyassociated on a naturally occurring chromosome.

The DNA may also be used to identify expression of the gene in abiological specimen. The manner in which one probes cells for thepresence of particular nucleotide sequences, as genomic DNA or RNA, iswell established in the literature and does not require elaborationhere. DNA or mRNA is isolated from a cell sample. The mRNA may beamplified by RT-PCR, using reverse transcriptase to form a complementaryDNA strand, followed by polymerase chain reaction amplification usingprimers specific for the subject DNA sequences. Alternatively, the mRNAsample is separated by gel electrophoresis, transferred to a suitablesupport, e.g. nitrocellulose, nylon, etc., and then probed with afragment of the subject DNA as a probe. Other techniques, such asoligonucleotide ligation assays, in situ hybridizations, andhybridization to DNA probes arrayed on a solid chip may also find use.Detection of mRNA hybridizing to the subject sequence is indicative ofBok gene expression in the sample.

The sequence of a Bok gene, including flanking promoter regions andcoding regions, may be mutated in various ways known in the art togenerate targeted changes in promoter strength, sequence of the encodedprotein, etc. The DNA sequence or protein product of such a mutationwill usually be substantially similar to the sequences provided herein,i.e. will differ by at least one nucleotide or amino acid, respectively,and may differ by at least two but not more than about ten nucleotidesor amino acids. The sequence changes may be substitutions, insertions ordeletions. Deletions may further include larger changes, such asdeletions of a domain or exon. Of particular interest is the creation ofBH3^(i) variants. Other modifications of interest include epitopetagging, e.g. with the FLAG system, HA, etc. For studies of subcellularlocalization, fusion proteins with green fluorescent proteins (GFP) maybe used.

Techniques for in vitro mutagenesis of cloned genes are known. Examplesof protocols for site specific mutagenesis may be found in Gustin etal., Biotechniques 14:22 (1993); Barany, Gene 37:111-23 (1985);Colicelli et al., Mol Gen Genet 199:537-9 (1985); and Prentki et al.,Gene 29:303-13 (1984). Methods for site specific mutagenesis can befound in Sambrook et al., Molecular Cloning: A Laboratory Manual, CSHPress 1989, pp. 15.3-15.108; Weiner et al., Gene 126:35-41 (1993);Sayers et al., Biotechniques 13:592-6 (1992); Jones and Winistorfer,Biotechniques 12:528-30 (1992); Barton et. al., Nucleic Acids Res18:7349-55 (1990); Marotti and Tomich, Gene Anal Tech 6:67-70 (1989);and Zhu, Anal Biochem 177:1204 (1989). Such mutated genes may be used tostudy structure-function relationships of Bok, or to alter properties ofthe protein that affect its function or regulation.

Other nucleic acids of the invention include pro-apoptotic BH3^(i)variants. These may be synthesized by using techniques of in vitromutagenesis and genetic engineering to inactivate the BH3 domain of Bokrelated genes. The wild-type sequence of these genes are known andpublically available, e.g. in Genbank the human Bak gene has theaccession no. U23765; human Bak-2 gene has the accession no. U16812;human Bik gene has the accession no. U34584; human Bax genes have theaccession nos. L22475, L22474 and L22473. One of skill in the art cangenerate the physical nucleic acid from the database sequence by variousmeans, e.g. synthesis of primers and PCR amplification, screening. cDNAlibraries, etc.

Bok Polypeptides

The subject nucleic acids may be employed for producing all or portionsof Bok polypeptides or BH3^(i) variants of pro-apoptotic Bok relatedpolypeptides. For expression, an expression cassette may be employed.The expression vector will provide a transcriptional and translationalinitiation region, which may be inducible or constitutive, where thecoding region is operably linked under the transcriptional control ofthe transcriptional initiation region, and a transcriptional andtranslational termination region. These control regions may be native toa Bok gene, or may be derived from exogenous sources.

The peptide may be expressed in prokaryotes or eukaryotes in accordancewith conventional ways, depending upon the purpose for expression. Forlarge scale production of the protein, a unicellular organism, such asE. coli, B. subtilis, S. cerevisiae, insect cells in combination withbaculovirus vectors, or cells of a higher organism such as vertebrates,particularly mammals, e.g. COS 7 cells, may be used as the expressionhost cells. In some situations, it is desirable to express the Bok genein eukaryotic cells, where the Bok protein will benefit from nativefolding and post-translational modifications. Small peptides can also besynthesized in the laboratory. Peptides that are subsets of the completeBok sequence, e.g. peptides of at least about 4 amino acids, usually atleast about 8 amino acids, more usually at least about 16 amino acids,up to and including functional domains, and the complete Bokpolypeptide, may be used to identify and investigate parts of theprotein important for function, or to raise antibodies directed againstthese regions.

With the availability of the protein or fragments thereof in largeamounts, by employing an expression host, the protein may be isolatedand purified in accordance with conventional ways. A lysate may beprepared of the expression host and the lysate purified using HPLC,exclusion chromatography, gel electrophoresis, affinity chromatography,or other purification technique. The purified protein will generally beat least about 80% pure, preferably at least about 90% pure, and may beup to and including 100% pure. Pure is intended to mean free of otherproteins, as well as cellular debris.

The expressed Bok polypeptides are useful for the production ofantibodies, where short fragments provide for antibodies specific forthe particular polypeptide, and larger fragments or the entire proteinallow for the production of antibodies over the surface of thepolypeptide. Epitopes for immunization may comprise one or more of theconserved BH domains. Antibodies may be raised to the wild-type orvariant forms of Bok. Antibodies may be raised to isolated peptidescorresponding to these domains, or to the native protein.

Antibodies are prepared in accordance with conventional ways, where theexpressed polypeptide or protein is used as an immunogen, by itself orconjugated to known immunogenic carriers, e.g. KLH, pre-S HBsAg, otherviral or eukaryotic proteins, or the like. Various adjuvants may beemployed, with a series of injections, as appropriate. For monoclonalantibodies, after one or more booster injections, the spleen isisolated, the lymphocytes immortalized by cell fusion, and then screenedfor high affinity antibody binding. The immortalized cells, i.e.hybridomas, producing the desired antibodies may then be expanded. Forfurther description, see Monoclonal Antibodies: A Laboratory Manual,Harlow and Lane eds., Cold Spring Harbor Laboratories, Cold SpringHarbor, N.Y., 1988. If desired, the mRNA encoding the heavy and lightchains may be isolated and mutagenized by cloning in E. coli, and theheavy and light chains mixed to further enhance the affinity of theantibody. Alternatives to in vivo immunization as a method of raisingantibodies include binding to phage “display” libraries, usually inconjunction with in vitro affinity maturation.

Modulation of Gene Expression

The Bok genes, gene fragments, or the encoded protein or proteinfragments, including BH3^(i) variants of related pro-apoptotic sequence,are useful in gene therapy to treat disorders associated with a deficitin pro-apoptosis proteins, including different states of tumorgenesis.This approach is also useful in treating proliferative conditions ofreproductive cells, such as uterine cell hyperplasia, leiomyoma andtumorigenesis. Expression vectors may be used to introduce the codingsequence into a cell. Such vectors generally have convenient restrictionsites located near the promoter sequence to provide for the insertion ofnucleic acid sequences. Transcription cassettes may be preparedcomprising a transcription initiation region, the target gene orfragment thereof, and a transcriptional termination region. Thetranscription cassettes may be introduced into a variety of vectors,e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and the like,where the vectors are able to transiently or stably be maintained in thecells, usually for a period of at least about one day, more usually fora period of at least about several days to several weeks.

The exogenous coding sequence or protein may be introduced into tissuesor host cells by any number of routes, including viral infection,microinjection, or fusion of vesicles. Methods that localize the agentto the particular targeted tissues are of interest.

Antisense molecules can be used to down-regulate expression of Bok incells. The anti-sense reagent may be antisense oligonucleotides (ODN),particularly synthetic ODN having chemical modifications from nativenucleic acids, or nucleic acid constructs that express such anti-sensemolecules as RNA. The antisense sequence is complementary to the mRNA ofthe targeted gene, and inhibits expression of the targeted geneproducts. Antisense molecules inhibit gene expression through variousmechanisms, e.g. by reducing the amount of mRNA available fortranslation, through activation of RNAse H, or steric hindrance. One ora combination of antisense molecules may be administered, where acombination may comprise multiple different sequences.

Antisense molecules may be produced by expression of all or a part ofthe target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996) Nature Biotechnology 1 4:840-844).

A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methodsknown in the art (see Wagner et al. (1993) supra. and Milligan et al.,supra.) Preferred oligonucleotides are chemically modified from thenative phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which alter thechemistry of the backbone, sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH2-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The α-anomer of deoxyribose may be used, where the base isinverted with respect to the natural β-anomer. The 2′-OH of the ribosesugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

Genetically Altered Cell or Animal Models for Bok Function

The subject nucleic acids can be used to generate transgenic animals orsite specific gene modifications in cell lines. Transgenic animals maybe made through homologous recombination, where the normal Bok locus isaltered. Alternatively, a nucleic acid construct is randomly integratedinto the genome. Vectors for stable integration include plasmids,retroviruses and other animal viruses, YACs, and the like.

The modified cells or animals are useful in the study of pro-apoptoticgene function and regulation. For example, a series of small deletionsand/or substitutions may be made in the Bok gene to determine the roleof different exons in oncogenesis, signal transduction, etc. Of interestare the use of Bok to construct transgenic animal models forproliferative disorders, e.g. endometriosis, where expression of Bok isspecifically reduced or absent. Specific constructs of interest includeanti-sense Bok, which will block Bok expression, expression of dominantnegative Bok mutations. A detectable marker, such as lac Z may beintroduced into the Bok locus, where upregulation of Bok expression willresult in an easily detected change in phenotype.

One may also provide for expression of the Bok gene or variants thereofin cells or tissues where it is not normally expressed or at abnormaltimes of development. By providing expression of Bok protein in cells inwhich it is not normally produced, one can induce changes in cellbehavior.

DNA constructs for homologous recombination will comprise at least aportion of the Bok gene with the desired genetic modification, and willinclude regions of homology to the target locus. Conveniently, markersfor positive and negative selection are included. Methods for generatingcells having targeted gene modifications through homologousrecombination are known in the art. For various techniques fortransfecting mammalian cells, see Keown et al. (1990) Methods inEnzymoloyy 185:527-537.

For embryonic stem (ES) cells, an ES cell line may be employed, orembryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of leukemia inhibitingfactor (LIF). When ES or embryonic cells have been transformed, they maybe used to produce transgenic animals. After transformation, the cellsare plated onto a feeder layer in an appropriate medium. Cellscontaining the construct may be detected by employing a selective medium. After sufficient time for colonies to grow, they are picked andanalyzed for the occurrence of homologous recombination or integrationof the construct. Those colonies that are positive may then be used forembryo manipulation and blastocyst injection. Blastocysts are obtainedfrom 4 to 6 week old superovulated females. The ES cells aretrypsinized, and the modified cells are injected into the blastocoel ofthe blastocyst. After injection; the blastocysts are returned to eachuterine horn of pseudopregnant females. Females are then allowed to goto term and the resulting offspring screened for the construct. Byproviding for a different phenotype of the blastocyst and thegenetically modified cells, chimeric progeny can be readily detected.

The chimeric animals are screened for the presence of the modified geneand males and females having the modification are mated to producehomozygous progeny. If the gene alterations cause lethality at somepoint in development, tissues or organs can be maintained as allogeneicor congenic grafts or transplants, or in in vitro culture. Thetransgenic animals may be any non-human mammal, such as laboratoryanimals, domestic animals, etc. The transgenic animals may be used infunctional studies, drug screening, etc.

In Vitro Models for Bok Function

The availability of a number of members in the bcl-2 gene family, aspreviously described, allows in vitro reconstruction of the apoptosispathway. Two or more of the components may be combined in vitro, and thebehavior assessed in terms of activation of transcription of specifictarget sequences; modification of protein components, e.g. proteolyticprocessing, phosphorylation, methylation, etc.; ability of differentprotein components to bind to each other, etc. The components may bemodified by sequence deletion, substitution, etc. to determine thefunctional role of specific domains.

Drug screening may be performed using an in vitro model, a geneticallyaltered cell or animal, or purified protein. One can identify ligands orsubstrates that bind to, modulate or mimic the action of Bok and otherpro-apoptotic proteins. Areas of investigation include the developmentof cancer treatments, agents that modulate Bok expression, etc.

Drug screening identifies agents that provide a replacement for Bokfunction in abnormal cells. Conversely, agents that reverse Bok functionmay stimulate controlled growth and healing. Of particular interest arescreening assays for agents that have a low toxicity for human cells. Awide variety of assays may be used for this purpose, including labeledin vitro protein-protein binding assays, electrophoretic mobility shiftassays, immunoassays for protein binding, and the like. The purifiedprotein may also be used for determination of three-dimensional crystalstructure, which can be used for modeling intermolecular interactions.

The term “agent” as used herein describes any molecule, e.g. protein orpharmaceutical, with the capability of altering or mimicking thephysiological function of Bok. Generally a plurality of assay mixturesare run in parallel with different agent concentrations to obtain adifferential response to the various concentrations. Typically, one ofthese concentrations serves as a negative control, i.e. at zeroconcentration or below the level of detection.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Where the screening assay is a binding assay, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Themixture of components are added in any order that provides for therequisite binding. Incubations are performed at any suitabletemperature, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Typically between 0.1 and 1 hours willbe sufficient.

Other assays of interest detect agents that mimic Bok function. Forexample, an expression construct comprising a Bok gene may be introducedinto a cell line under conditions that allow expression. The level ofBok activity is determined by a functional assay. In one screeningassay, candidate agents are added, and the ability to down-regulate itsactivity is detected. In another assay, the ability of candidate agentsto enhance Bok function is determined. Alternatively, candidate agentsare added to a cell that lacks functional Bok, and screened for theability to reproduce Bok in a functional assay.

The compounds having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a host fortreatment of proliferative diseases, etc. The compounds may also be usedto enhance Bok function. The inhibitory agents may be administered in avariety of ways, orally, topically, parenterally e.g. subcutaneously,intraperitoneally, by viral infection, intravascularly, etc. Topicaltreatments are of particular interest. Depending upon the manner ofintroduction, the compounds may be formulated in a variety of ways. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1-100 wt. %.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

Diagnostic Uses

The subject nucleic acid and/or polypeptide compositions may be used toanalyze a patient sample for the presence of polymorphisms associatedwith a disease state or genetic predisposition to a disease state.Biochemical studies may be performed to determine whether a sequencepolymorphism in a Bok coding region or control regions is associatedwith disease. Disease associated polymorphisms may include deletion ortruncation of the gene, mutations that alter expression level, etc.

Changes in the promoter or enhancer sequence that may affect expressionlevels of Bok can be compared to expression levels of the normal alleleby various methods known in the art. Methods for determining promoter orenhancer strength include quantitation of the expressed natural protein;insertion of the variant control element into a vector with a reportergene such as β-galactosidase, luciferase, chloramphenicolacetyltransferase, etc. that provides for convenient quantitation; andthe like.

A number of methods are available for analyzing nucleic acids for thepresence of a specific sequence, e.g. a disease associated polymorphism.Where large amounts of DNA are available, genomic DNA is used directly.Alternatively, the region of interest is cloned into a suitable vectorand grown in sufficient quantity for analysis. Cells that express Bokmay be used as a source of mRNA, which may be assayed directly orreverse transcribed into cDNA for analysis. The nucleic acid may beamplified by conventional techniques, such as the polymerase chainreaction (PCR), to provide sufficient amounts for analysis. The use ofthe polymerase chain reaction is described in Saiki, et al. (1985)Science 239:487, and a review of techniques may be found in Sambrook, etal. Molecular Cloning: A Laboratory Manual, CSH Press 1989,pp.14.2-14.33. Alternatively, various methods are known in the art thatutilize oligonucleotide ligation as a means of detecting polymorphisms,for examples see Riley et al. (1990) N.A.R. 18:2887-2890; and Delahuntyet al. (1996) Am. J. Hum. Genet. 58:1239-1246.

A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

The sample nucleic acid, e.g. amplified or cloned fragment, is analyzedby one of a number of methods known in the art. The nucleic acid may besequenced by dideoxy or other methods, and the sequence of basescompared to a wild-type Bok sequence. Hybridization with the variantsequence may also be used to determine its presence, by Southern blots,dot blots, etc. The hybridization pattern of a control and variantsequence to an array of oligonucleotide probes immobilised on a solidsupport, as described in U.S. Pat. No. 5,445,934, or in WO95/35505, mayalso be used as a means of detecting the presence of variant sequences.Single strand conformational polymorphism (SSCP) analysis, denaturinggradient gel electrophoresis (DGGE), and heteroduplex analysis in gelmatrices are used to detect conformational changes created by DNAsequence variation as alterations in electrophoretic mobility.Alternatively, where a polymorphism creates or destroys a recognitionsite for a restriction endonuclease, the sample is digested with thatendonuclease, and the products size fractionated to determine whetherthe fragment was digested. Fractionation is performed by gel orcapillary electrophoresis, particularly acrylamide or agarose gels.

Screening for mutations in Bok may be based on the functional orantigenic characteristics of the protein. Protein truncation assays areuseful in detecting deletions that may affect the biological activity ofthe protein. Various immunoassays designed to detect polymorphisms inBok proteins may be used in screening. Where many diverse geneticmutations lead to a particular disease phenotype, functional proteinassays have proven to be effective screening tools. The activity of theencoded Bok protein may be determined by comparison with the wild-typeprotein.

Antibodies specific for a Bok may be used in staining or inimmunoassays. Samples, as used herein, include biological fluids such assemen, blood, cerebrospinal fluid, tears, saliva, lymph, dialysis fluidand the like; organ or tissue culture derived fluids; and fluidsextracted from physiological tissues. Also included in the term arederivatives and fractions of such fluids. The cells may be dissociated,in the case of solid tissues, or tissue sections may be analyzed.Alternatively a lysate of the cells may be prepared.

Diagnosis may be performed by a number of methods to determine theabsence or presence or altered amounts of normal or abnormal Bok inpatient cells. For example, detection may utilize staining of cells orhistological sections, performed in accordance with conventionalmethods. Cells are permeabilized to stain cytoplasmic molecules. Theantibodies of interest are added to the cell sample, and incubated for aperiod of time sufficient to allow binding to the epitope, usually atleast about 10 minutes. The antibody may be labeled with radioisotopes,enzymes, fluorescers, chemiluminescers, or other labels for directdetection. Alternatively, a second stage antibody or reagent is used toamplify the signal. Such reagents are well known in the art. Forexample, the primary antibody may be conjugated to biotin, withhorseradish peroxidase-conjugated avidin added as a second stagereagent. Alternatively, the secondary antibody conjugated to aflourescent compound, e.g. flourescein, rhodamine, Texas red, etc. Finaldetection uses a substrate that undergoes a color change in the presenceof the peroxidase. The absence or presence of antibody binding may bedetermined by various methods, including flow cytometry of dissociatedcells, microscopy, radiography, scintillation counting, etc.

Diagnostic screening may also be performed for polymorphisms that aregenetically linked to a disease predisposition, particularly through theuse of microsatellite markers or single nucleotide polymorphisms.Frequently the microsatellite polymorphism itself is not phenotypicallyexpressed, but is linked to sequences that result in a diseasepredisposition. However, in some cases the microsatellite sequenceitself may affect gene expression. Microsatellite linkage analysis maybe performed alone, or in combination with direct detection ofpolymorphisms, as described above. The use of microsatellite markers forgenotyping is well documented. For examples, see Mansfield et al. (1994)Genomics 24:225-233; Ziegle et al. (1992) Genomics 14:1026-1031; Dib etal., supra.

It is to be understood that this invention is not limited to theparticular methodology, protocols, formulations and reagents described,as such may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acomplex” includes a plurality of such complexes and reference to “theformulation” includes reference to one or more formulations andequivalents thereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications, mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing, for example, the methods,ligands, and methodologies that are described in the publications whichmight be used in connection with the presently described invention. Thepublications discussed above and throughout the text are provided solelyfor their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior invention.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight, andpressure is at or near atmospheric.

Experimental EXAMPLE 1 Isolation and Characterization of Bok cDNA

Materials and Methods

Two-hybrid screening. The full-length open reading frame (ORF) of ratMcl-1 cDNA was fused in frame with the GAL4-binding domain (GAL4-BD)into the pGBT-9 yeast shuttle vector (Clontech, Palo Alto, Calif.). Thisvector was used to identify Mcl-1-interacting proteins by screening 1.5million transformants from a GAL4-activation domain (AD)-tagged ovarianfusion Matchmaker cDNA library. The ovarian cDNAs were prepared from27-day-old Sprague-Dawley rats primed for 36 h with 10 IU equinegonadotropin. Yeast cells were first transformed with pGBT9-Mcl-1 andcolonies selected in plates deficient for tryptophan. In the secondstep, cells were transformed with cDNAs from the ovarian library beforeselection of clones in plates lacking tryptophan, leucine and histidine.Positive transformants were further selected for growth in mediacontaining 5 mM 3-aminotriazole. Individual AD-fusion cDNAs wereretrieved following transformation of E. coli cells.

A total of 40 potential Mcl-1-interacting clones were re-screenedagainst the empty vector or vector encoding different Bcl-2 proteins toeliminate false positives. Three clones of Bok cDNAs were isolated basedon their ability to interact with Mcl-1 in an HF7c yeast reporter strain(Fields & Song (1989) Nature 340, 245-247). DNA sequence analysis andcomparison with known genes using the BLASTX algorithm revealed that thepositive clones encode a polypeptide sharing high homology with Bcl-2proteins. Further analysis of expressed sequence tags (EST) in theGenBank revealed that EST accession number AA103989 has greater than 98%identity with the 5′-sequence of cloned cDNA and contains extra5′-sequence of the murine Bok homolog. Full-length ORF and5′-untranslated sequence of rat Bok were obtained by PCR using theGAL4-AD-tagged ovarian Matchmaker cDNA library as the template and anupstream primer based on murine EST. Complementary DNA fragments with anidentical ORF were also obtained in separate PCR using a rat brain cDNAlibrary (Stratagene, La Jolla, Calif.) as the template. Interactionsbetween Bok and different Bcl-2 members were assessed in the yeasttwo-hybrid system using pGBT9 GAL4-BD and pGADGH GAL4-AD vectors (Bartelet al. (1993) in Cellular Interaction in Development: A PracticalApproach, ed. Hartley, D. A. (Oxford University Press, Oxford), pp.153-179). Specific binding of different protein pairs was evaluatedbased on the activation of GAL1-HIS3 and GAL4-lacZ reporter genes.

Cell culture and transfection with plasmids. For the expression of Bcl-2proteins in eukaryotic cells, PCR-generated ORF of different cDNAs weresubcloned into the pcDNA3 vector (Invitrogen, Inc., San Diego, Calif.).Following transfection of cDNAs, cell death was monitored (Kumar et al.(1994) Genes Dev. 8, 1613-1626). CHO cells (2×10⁵/well) were cultured inDulbecco's modified Eagle's medium (DMEM)/F12 supplemented with 10%fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mMglutamine. One day later, cells were transfected using the lipofectamineprocedure (Life Technologies, Gaithersburg, Md.) with the empty pcDNA3expression vector or the same vector containing different cDNAs,together with {fraction (1/10)}-{fraction (1/20)} fractions of anindicator plasmid pCMV-β-gal to allow the identification of transfectedcells. Inclusion of 10- to 20-fold excess of expression vectors ascompared to the pCMV-β-gal reporter plasmid ensured that most of theβ-galactosidase expressing cells also expressed the protein(s) underinvestigation. Cells were incubated with plasmids in a serum-free mediumfor 4 h, followed by adding fetal bovine serum to a final concentrationof 5% and further incubation for 14 h. After an additional culture infresh medium for 18 h, cells were fixed by 0.25% glutaraldehyde andstained with X-gal (0.4 mg/ml) to detect β-galactosidase expression. Thenumber of blue cells was counted by microscopic examination (Kumar etal. (1994) Genes Dev. 8, 1613-1626). To verify the nature of cell death,total cellular DNA was extracted for 3′-end labeling of DNA ends at 18 hafter transfection with ³²P-ddATP before gel fractionation to identifyinternucleosomal DNA fragmentation (Hsu et al. (1996) Endocrinology 137,4837-4843). Statistical differences among treatment groups were analyzedusing one-way ANOVA and Scheffe F-test.

Northern and Southern blots and in situ analyses For Northern blotanalysis of Bok expression, tissues were collected from 27-day-oldSprague-Dawley rats (Simonsen Lab, Gilroy, Calif.). For Bok expressionin ovarian cells, ovaries were obtained from 26-day-old rats implantedfor two days with a diethylstilbestrol capsule to stimulate developmentof multiple early antral follicles (Bicsak et al. (1986) Endocrinology119, 2711-2719). Granulosa cells were prepared by needle puncture. Forthe extraction of total RNA, tissues were homogenized in Tri-Reagentsolution (Molecular Research Center, Inc., Cincinnati, Ohio) and atleast two pools from each treatment group were used. In addition, poly(A+) RNA was isolated using the Oligotex oligo-dT resin (Qiagen, Inc.,Chatsworth, Calif.). Aliquots of each sample were denatured andfractionated in 1% agarose gels containing formaldehyde before northernblotting analysis. Membranes were pre-hybridized for 4 h at 65° C. in asolution containing 50% formamide, 5×sodium phosphate buffer (SSPE),5×Denhardt's solution, 0.5% SDS and 500 μg/ml yeast tRNA. This wasfollowed by overnight hybridization in the same conditions but with1×10⁶ cpm/ml of ³²P-labeled Bok or GAPDH cRNA probe. Afterhybridization, the membranes were washed twice in 2×SSC, 1% SDS at roomtemperature, followed by two washes in 0.1×SSC, 1% SDS at 65° C. beforeexposure to Kodak RX films (Eastman Kodak, Rochester, N.Y.). For studieson the conservation of the Bok gene during evolution, the Zoo blot(Clontech) containing genomic DNA from different vertebrate species washybridized with a ³²P-labeled rat Bok cDNA probe under stringentconditions.

For in situ hybridization analysis of Bok mRNA expression, ovaries from26-day-old rats were isolated and fixed at 4° C. for 4 h in 4%paraformaldehyde in PBS (pH 7.4), followed by overnight dehydration in0.5M sucrose. Tissue blocks were embedded in Tissue-Tek solution (SakuraFinetek USA Inc., Torrence, Calif.) and snapped frozen in liquidnitrogen. Twelve μm thick cryo-sections were mounted on chargedmicroscopic slides (Fischer Scientific, Pittsburgh, Pa.), post-fixed in4% paraformaldehyde and stored at −70 C. for up to 1 month.Hybridization and washes of cryosections were as previously described(Hsu et al, supra.). After two weeks of exposure under NTB2 emulsion(Kodak), the slides were developed, counter-stained and mounted withPermount (Fisher Scientific, Fair Lawn, N.J.) for observation andphotography using a Nikon Optiphot microscope.

Results

Using the anti-apoptotic protein Mcl-1 as bait, we screened an ovarianfusion cDNA library and isolated three Bok clones. Subsequent DNAsequencing and identification of homologous murine ESTs allowedisolation of full-length Bok cDNAs following PCR of ovarian and braincDNA libraries. The ORF of Bok encoded a protein of 213 amino acidsshowing no identity with any known gene. The novel protein has apredicted molecular weight of 23.5 kD and a pl of 9.1. The methionineinitiation codon conformed to the consensus Kozak sequence andhydrophobicity analysis predicted the presence of a C-terminaltransmembrane domain. In addition, two potential phosphorylation siteswere found near the N-terminal region. Comparison of DNA sequences amongdifferent Bcl-2 proteins indicated that Bok was a novel member of thisfamily showing conserved BH 1, 2 and 3 domains. However, the BH4 domain,known to be important for the anti-apoptotic function of mammalian Bcl-2proteins, was missing in Bok. Closer comparison indicated the core BH1domain of Bok (TWGK) was less conserved as compared with other Bcl-2proteins (NWGR). For the BH3 domain found in pro-apoptotic members, thecore sequence (GDE) was conserved in Bok but the flanking sequences weredifferent. Furthermore, analysis of the phylogenetic relatedness of thedifferent Bcl-2 members suggests that, during evolution, Bok divergedearly from other Bcl-2 proteins.

We investigated interactions between Bok and different anti- andpro-apoptotic Bcl-2 proteins. Bok only interacts with selectiveanti-apoptotic Bcl-2 proteins in the yeast two-hybrid system. Yeastcells were grown in the selective media containing 5 mM 3-aminotriazoleand without Trp, Leu and His. Prominent growth of yeast coloniesexpressing Bok fused to the GAL4. activation domain together with Mcl-1,BHRF1 or Bfl-1 fused to the GAL4 binding domain could be seen. Minimalgrowth of yeast colonies was found in cells that express the same Bokexpressing vector together with Bcl-2, Bcl-xL, Bcl-w, BAD, Bax, Bak orBlk fused to the GAL4 binding domain. In addition, prominent growth ofcolonies expressing Bcl-xL and different pro-apoptotic Bcl-2 proteinsindicated that the lack of growth in yeast cells expressing Bok anddifferent pro-apoptotic family members was not due to suppression ofcell growth by these pro-apoptotic proteins.

Of interest, Bok did not interact with any pro-apoptotic members tested.To demonstrate that the lack of interactions between Bok andpro-apoptotic Bcl-2 proteins was not due to the killing of yeast cellsby these apoptosis agonists, we also tested the growth of yeast cellsco-transformed with Bcl-xL and different pro-apoptotic proteins.Although Bcl-xL showed negligible interaction with Bok, it interactedstrongly with all the pro-apoptotic members tested.

To further study the restricted dimerization property of Bok withselective anti-apoptotic proteins, we tested the growth of yeast cellsthat were co-transformed with different pairs of pro- and anti-apoptoticBcl-2 proteins. Several pro-apoptotic proteins (Bak, Bik and Bax),unlike Bok, all interacted strongly with diverse anti-apoptotic proteinstested, data shown in Table 1, suggesting the restrictedhetero-dimerization property of Bok was unique.

TABLE 1 pGBT-9 BHRF1 Mcl-1 Bfl-1 Bcl-2 Bcl-xL Bcl-w Bok −− ++ ++ + −− −−−− Bax −− ++ ++ ++ ++ ++ ++ Bak −− ++ ++ ++ ++ ++ ++ Bik −− ++ ++ ++ ++++ ++ Summary of protein-protein interactions between pairs ofpro-apoptotic proteins (Bok, Bak, Bik and Bax) and differentanti-apoptotic Bcl-2 members. The positive signs indicate prominent (++)or moderate (+) yeast cell growth whereas the negative signs (−)indicate the absence of reporter gene expression.

The ability of Bok to regulate apoptosis in mammalian cells wasinvestigated. In CHO cells, transfection with expression vectorsencoding Bok for 36 h induced cell death. The pro-apoptotic effect ofBok was specific because transfection of the empty plasmid or the sameplasmid containing Bok cDNA in reverse orientation did not affect cellsurvival. Furthermore, co-expression of P35, a cysteine proteaseinhibitor derived from the baculovirus (Bump et al. (1995) Science 269,1885-1888), prevented Bok-induced cell killing as indicated by increasesin the number of viable cells.

Normal cell morphology was found in cells transiently transfected withthe empty pcDNA3 expression vector (2.1 μg DNA/35 mm dish) or the vectorcontaining Bok cDNA in reverse orientation. Cells were also transfectedwith the Bok expression vector without or with an equal amount of theP35-expressing construct.

The 3′-end labeling of genomic DNA fragments at an earlier time point(18 h) further demonstrated the induction of internucleosomal DNAfragmentation following Bok over-expression, confirming the induction ofapoptosis. The observed DNA fragmentation was blocked by co-expressionwith P35. CHO cells were treated as described in above. At 18 h aftertransfection, cellular DNA was extracted for analysis of DNAfragmentation using a 3′-end labeling method.

Quantitative analysis also indicated that Bok over-expression decreasedviable cell number by 75% whereas co-expression of P35 completelyreversed Bok killing (FIG. 1), substantiating the involvement ofcaspases in Bok action.

We further tested if the restricted hetero-dimerization of Bok withselective anti-apoptotic Bcl-2 members found in the yeast two-hybridsystem could be substantiated in mammalian cells. Bok was co-expressedwith Mcl-1, BHRF1 or Bcl-2 in CHO cells. As shown in FIG. 2, Bok-inducedapoptosis was attenuated following co-expression with Mcl-1 or BHRF1;but co-expression with Bcl-2 was ineffective in blocking Bok action.Transfection of the same Bcl-2 expression vector was, however, capableof blocking apoptosis induced by staurosporin.

The expression of Bok mRNA in diverse rat tissues was examined. Highlevels of Bok transcript of ˜1.5 kb in size were abundant in the ovary,testis and uterus, less abundant in the brain, heart and intestine andnegligible in other tissues examined. Further analysis of Bok mRNA inisolated granulosa cells demonstrate high levels of expression in thesecells that undergo apoptosis during follicle degeneration. In situhybridization analyses further confirmed high levels of the Boktranscript in the granulosa cells of antral and preantral follicles,with minimal signals in theca and interstitial cells

For northern blot analysis, poly (A)+-selected RNA from differenttissues of rats at 27 days of age or from isolated granulosa cells ofestrogen-treated rats were hybridized with a ³²P-labeled Bok cRNA probe.After washing, the blots were exposed to X-ray films at −70 C. for fivedays. Subsequent hybridization with a GAPDH cRNA probe was performed toestimate nucleic acid loading (8 h exposure). For in situ hybridizationanalysis, ovaries from immature eCG-treated rats were probed with theanti-sense Bok cRNA. Positive signals were found in granulosa cells ofpreantral follicles and an antral follicle. No signal was found in asection hybridized with the sense Bok probe.

Conservation of the Bok gene in diverse vertebrates was tested usingSouthern blot hybridization of genomic DNA from different species. DNAwas digested with the EcoRI enzyme and probed with a Bok cDNA probe.Following hybridization at 68 C., the membrane was washed under highstringency conditions (1% SDS, 0.1×SSC at 65 C.) before exposure. Underhigh stringency washing conditions, the rat cDNA cross-hybridizedstrongly with rat, human and monkey genomic DNA and weakly with dog, cowand rabbit DNA. Negligible hybridization signals were found for chickenDNA.

A cDNA encoding the human bok gene was isolated from a human ovary cDNAlibrary. A partial sequence of the human protein is provided in SEQ IDNO:3.

Discussion

A new pro-apoptotic Bcl-2-related protein Bok has been identified basedon its binding to an ovarian anti-apoptosis protein Mcl-1. In additionto its elevated expression in several reproductive tissues, Bok wasfound in diverse other tissues. In addition, Bok shows a selectivehetero-dimerization property by interacting with some (Mcl-1, BHRF1 andBfl-1) but not other (Bcl-2, Bcl-xL and Bcl-w) anti-apoptotic proteins.Coupled with findings that Bok-induced apoptosis could only beantagonized by selective anti-apoptotic proteins, the present datasuggest that different pro- and anti-apoptotic Bcl-2 protein pairs mayplay tissue-specific roles in the regulation of apoptosis. Due to thehigher expression of Bok to ovarian granulosa cells and severalreproductive tissues characterized by hormonally regulated cyclic cellturnover, further analyses of Bok action in the gonads and uterus couldprovide unique models to study the hormonal regulation of apoptosis.Because most of the Bcl-2-related proteins have been identified in thelymphoid system, the present yeast two-hybrid screen provides anexperimental paradigm to isolate novel Bcl-2. homologs essential forapoptosis regulation in other tissues.

Although the mechanism by which the Bcl-2 proteins participates in the“decision” step of apoptosis is not clear, the ratio of anti- andpro-apoptotic Bcl-2 members and their hetero- and homo-dimerization arebelieved to determine whether a cell will respond to an apoptoticsignal. Among the Bcl-2 family of proteins, several homology domainshave been found to be essential for their function. Bok containsconserved BH1, 2 and 3 domains but lacks the BH4 domain found in mostanti-apoptotic members. In addition, the conserved NH1 region importantfor the survival function of several anti-apoptotic Bcl-2 proteins isalso absent in Bok. Consistent with its structural features,over-expression of Bok in CHO cells induces apoptosis based on observedcell morphology and internucleosomal DNA fragmentation. Bok-induced cellkilling, like that induced by Bax and BAD, is mediated by caspases asdemonstrated by the suppressive actions of the baculoviral P35 protein.

Among the pro-apoptotic Bcl-2 proteins, Bok is most similar to Bax andBak in having the BH1, 2 and 3 domains plus the C-terminal transmembranesequence. Studies on Bax and Bak with truncation in different BH domainssuggested that these pro-apoptotic proteins might exert their effectsby-hetero-dimerizing with Bcl-2 or Bcl-xL. Competitive dimerizationbetween selective pairs of anti- and pro-apoptotic Bcl-2 proteins isbelieved to be involved in the “decision” step of apoptosis.Furthermore, interactions among the Bcl-2 family of proteins appear toexhibit a defined selectivity and hierarchy. For example, theanti-apoptotic E1B protein shows preferential binding to pro-apoptoticBcl-2 proteins, whereas the pro-apoptotic Hrk binds only to theanti-apoptotic family member. Thus, the pro-apoptotic protein Bok mayregulate apoptosis through similar mechanisms by forming hetero-dimerswith selective anti-apoptotic proteins.

Analysis of the relatedness of amino acid sequences of different Bcl-2proteins indicated that Bok is not closely related to any particularBcl-2 member and probably diverged early during evolution. The lessconserved BH1 domain of Bok may determine its unique hetero-dimerizationproperty. In both yeast and mammalian cells, Bok interacts with some butnot other anti-apoptotic proteins, suggesting the possible evolution ofselective pairs of death agonists and antagonists with restrictedhetero-dimerization properties to confer tissue specificity of the deathprogram. Apoptosis induced by Bok in transfected CHO cells could bemediated through inhibition of the protection afforded by Mcl-1 or otherBok partners. It is likely that Bok may interact with its dimerizationpartner(s) including Mcl-1 and Bfl-1 in reproductive tissues to regulateapoptosis. Of interest, our recent data indicated that Mcl-1, but notBcl-2, is highly expressed in ovarian cells. Based on the suppression ofBok-induced apoptosis by BHRF1, it is possible that reproductive tissuesexpressing Bok are potential targets for this anti-apoptotic proteinencoded by the Epstein-Barr virus. Recent studies have suggested thatanti-apoptotic proteins may bind to ced4/Apaf-1 homologs, which, inturn, activate downstream caspases. Elucidation of thehetero-dimerization partner(s) for Bok in gonads and uterus would allowcharacterization of the putative ced4 homologs in these tissues.

Recent crystallographic analyses of complexes formed between theanti-apoptotic protein Bcl-xL and the BH3 domain of the pro-apoptoticBak indicated that the α-helix in the BH3 domain of different Bcl-2proteins plays a central role in defining the binding specificity toBcl- xL. Because Bok does not interact with Bcl-xL in the yeasttwo-hybrid system, further studies on the BH3 region of Bok and relatedproteins could define the specificity of hetero-dimerization amongdifferent pro- and anti-apoptotic protein pairs and their role inapoptosis regulation.

Although over-expression of Bax and Bak induces yeast cell death, thepresent Bok fusion protein did not affect yeast cell survival. Inaddition, lack of interactions between Bok and different pro-apoptoticBcl-2 proteins in the two-hybrid assay are not due to detrimentaleffects of these death agonists on yeast cells because co-transformationof these apoptosis agonists with Bcl-xL led to activation of thereporter genes. It is likely that moderate expression of these deathagonists using the present expression vector may not significantlyaffect yeast cell survival, thus allowing studies on interactionsbetween different Bcl-2 proteins.

The majority of ovarian follicles and about 50% of testicular germ cellsundergo apoptosis under normal physiological conditions, whereas themenstruation involves monthly apoptosis of uterine endometrial cells.The restricted expression of Bok in the gonads and uterus suggests itspotential role in the regulation of apoptosis in these tissues. It islikely that selective pairs of Bcl-2 agonists/antagonists may playtissue-specific roles in the regulation of apoptosis. Indeed, mutantmice deficient in Bcl-2 or Bax showed abnormality in apoptosisregulation only in distinct cell lineage. Although Bax-deficient miceshowed an accumulation of granulosa cells in atretic follicles, thesecells were still apoptotic, suggesting the involvement of additionalpro-apoptotic factors during ovarian follicle atresia. Because thepro-apoptotic protein Bax has been suggested to function as a tumorsuppressor gene in colon adenocarcinomas and because inactivation of Baxin transgenic mice leads to enhanced tumorigenesis, it would also beinteresting to investigate changes in Bok function during gonadal anduterine tumorigenesis. Because cyclic variations in reproductivehormones are essential in the regulation of apoptosis in gonadal anduterine tissues, future investigations on the hormonal regulation of theBok and its dimerization partner(s) in these reproductive tissues wouldallow the design of novel strategies to modulate reproductive functions.These studies could also provide understanding on the role of Bok ingonadal and uterine diseases associated with aberrant regulation ofapoptosis

EXAMPLE 2

Characterization of a Bok splicing variant with a truncated BH3 domain,which induces apoptosis but does not dimerize with anti-apoptotic Bcl-2proteins in vitro

A Bok splicing variant is identified in which the region encoded by exonthree is absent, creating a truncated short form (Bok-S) of thefull-length Bok protein (Bok-L). The skipping of exon three maintainsthe original reading frame and retains the BH2 and the C-terminalmembrane anchoring domains; however, parts of the BH3 and BH1 domainswere deleted. Functional analysis indicated that Bok-S is still capableof inducing apoptosis. The truncated Bok has lost its ability toheterodimerize with Mcl-1, BHRF-1 and Bfl-1, suggesting that theproapoptotic activity of this variant is not mediated by its binding toantiapoptotic Bcl-2 proteins.

Materials and Methods

Reverse transcription-PCR of the Bok-S transcript. Total RNA fromdifferent tissues was isolated from 27-day-old Sprague-Dawley rats usingan anion exchange resin chromatographic column (Qiagen, Chatsworth,Calif.) before reverse transcription with oligo (dT) 18 as primer in areaction containing RNase H-free reverse transcriptase from Moloneymurine leukemia virus (Clontech, Palo Alto, Calif.). For PCRamplification of Bok cDNAs, aliquots of DNA equivalent to 0.5 μg totalRNA were used in each reaction (50 μl). To minimize contamination duringPCR, control reactions containing a single primer or RNA without reversetranscriptase were routinely performed. All PCR was carried out underhigh stringency conditions (94° C., 45 s, 68° C. 45 s, 72° C. 4 min) for30 cycles.

Isolation of Bok genomic DNA, Southern blot hybridization and genomicanalysis. A genomic DNA fragment was isolated from a mouse BAC genomicDNA library (Genome Systems Inc., St. Louis, Mo.) using the full-lengthBok cDNA probe. The Bok genomic fragment was first analyzed byrestriction enzyme mapping, followed by subcloning into the pUC18 vectorfor dideoxy sequencing analysis of both DNA strands. Overlapping cloneswere isolated to define the direction of individual clones and tofacilitate assignment of intron-exon junctions. For Southern blothybridization analysis, genomic DNA (10 μg) was digested with indicatedrestriction enzymes, separated by electrophoresis on a 0.8% Agarose gel,and then transferred onto a Nylon membrane (Hybond-N, Amersham Corp.,Arlington Heights, Ill.). Hybridization was performed in the QuickHybbuffer (Clontech) at 60 C. with ³²P-labeled cDNA probes. The filterswere washed with 0.1×SSC and 0.5% SDS at 65 C. before exposure.

Generation of mutant constructs. Specific mutations in the BH3 domain ofBok-L were generated by a two-step PCR mutagenesis method using a BokcDNA template as previously described (Hsu et al. (1997) Mol Endocrinol11:1858-1867). The resulting PCR products were evaluated for correctsize on a 1% Agarose gel, purified, and subcloned into the EcoR1 site ofthe pcDNA3 vector for mammalian cell expression (Invitrogen, Inc., SanDiego, Calif.). Truncated Bax and Bak constructs (Bax-S and Bak-S) withhomologous deletion of the BH3-BH1 region found in Bok-S were alsogenerated using two-step overlapping PCR, and subcloned into the EcoRVsite of the pcDNA3 vector for eukaryotic cell expression (Hsu et al.supra.) For the yeast two-hybrid assay, mutant Bok-L cDNAs weresubcloned into the pGADGH expression vector. Restriction mapping anddideoxy sequencing confirmed proper orientation and the authenticity ofthe inserts.

Yeast two-hybrid assay. To study dimerization between different Bcl-2family proteins and variants or mutants of Bok, cDNAs for Bok-L, Bok-Sor Bok mutants were fused to the activation domain (AD) of GAL4 in ayeast shuttle vector pGADGH. Complementary DNAs encoding different Bcl-2proteins were fused to the GAL4-binding domain (BD) of pGBT9. Aftertransformation of yeast cells, colonies containing different proteinpairs were selected on plates lacking tryptophan and leucine. To testfor specific protein-protein interactions, positive transformants werefurther selected for growth in media deficient for tryptophan, leucineand histidine but containing 5-30 mM 3-aminotriazole to inhibitendogenous histidine production. A minimum of five independenttransformants containing each pair of fusion cDNAs was routinelyanalyzed.

Analysis of apoptosis in transfected CHO cells. Apoptosis was monitoredfollowing transfection of different cDNAs as previously described ( Hsuet al. (1997) Proc. Natl. Acad. Sci. USA 94:12401-12406). CHO cells(2×10⁵ cells/well) were cultured in Dulbecco's modified Eagle's medium(DMEM)/F12 supplemented with 10% fetal bovine serum, 100 U/mlpenicillin, 100 g/ml streptomycin and 2 mM glutamine. One day later,cells were transfected using the lipofectamine procedure (LifeTechnologies, Gaithersburg, Md.) with the pcDNA3 expression vector withor without different cDNA inserts, together with 1/10 to 1/20 amounts ofan indicator plasmid pCMV-β-gal to allow the identification oftransfected cells. Inclusion of a 10-fold excess of expression vectorsas compared with the pCMV-β-gal reporter plasmid ensured that most ofthe β-galactosidase-expressing cells also expressed the protein(s) underinvestigation. Cells were incubated with plasmids in a serum-free mediumfor 4 h, followed by the addition of fetal bovine serum to a finalconcentration of 5% and further incubation for 14 h. After an additionalculture in fresh medium for 18 h, cells were fixed by using 0.25%glutaraldehyde and stained with X-gal to detect β-galactosidaseexpression. The number of blue cells was counted by microscopicexamination. Data are expressed as the percentage (mean +/− SEM) ofviable cells as compared to the control group.

In vitro direct protein-binding assay. To further demonstrate thespecificity of interactions between Bok variants and antiapoptoticproteins, direct protein-protein interactions were studied usingrecombinant Bok and FLAG-tagged BHRF-1 translated in vitro.³⁵S-methionine labeled or nonlabeled proteins were generated using theTNT coupled reticulocyte lysate system (Promega, Madison, Wis.). Pairsof proteins were incubated in the binding buffer (PBS, 0.2% NP-40 andprotease inhibitor cocktail; Sigma, St. Louis, Mo.) for 2 h at 4° C.followed by incubation with 1.5 μg of M2 antibody against the FLAG tag(Kodak, Rochester, N.Y.) under gentle agitation. The complexes formedbetween the antibody and recombinant proteins were precipitated withProtein A Sepharose (Pharmacia Biotech, Uppsala, Sweden) and resolvedusing 12-15% SDS PAGE. Following fixation, gels were treated withAmplify fluorographic agents (Amersham Life Science Inc., England)before exposure to x-ray films.

Statistical analyses and sequence analysis. One-way analysis of variancefollowed by Scheffe's F-test was used to determine the statisticalsignificance of cell viability employing the STATVIEW software (AbacusConcepts, Inc., Berkeley, Calif.). The hydropathicity of proteinsequence was analyzed using Biology Workbench version 2.0(http://biology.ncsa.uiuc.edu/BW/BW.cgi).

Results

Existence of long and short Bok splicing variants in reproductivetissues. We amplified Bok cDNA from a rat ovarian cDNA library usingprimers flanking the open reading frame (ORF) of Bok. A 513 bp PCRproduct was obtained in addition to the predicted 642 bp band. DNAsequencing of the lower molecular weight product indicated it wasidentical to that of the Bok cDNA except that nucleotides encoding aminoacid 76-118 were missing. This short transcript (Bok-S) encoded a 170amino acid polypeptide and the deletion of 43 amino acids from thefull-length 213 amino acid protein (Bok-L) led to the fusion of theN-terminal half of the BH3 domain to the C-terminal half of the BH1domain. To confirm the authenticity of this variant, reversetranscription-PCR was performed using total RNA from different tissues.Electrophoresis analysis confirmed the presence of a PCR product of 642bp in the ovary, uterus and testis, tissues known to express the Boktranscript. In addition, a lower band of 513 bp was found in the ovary,less in the uterus and absent from the testis. Negative controlreactions using only a single primer or RNA without reversetranscription did not generate any products. Subsequent subcloning andsequencing confirmed that the 642 bp and 513 bp bands encode theexpected Bok-L and Bok-S transcripts, respectively.

The deduced amino acid sequence of Bok-S showed that a presumptivealternative splicing led to the disruption of both BH1 and BH3 domainsof Bok-L, changing the original BH3 sequence [SEQ ID NO:9] 71LLRLGDELEQIR 82 to [SEQ ID NO:10] 71 LLRLGITWGKW 82. However,Kyte-Doolittle hydropathicity analysis suggested that the hydropathicityprofile of the BH3/BH1 fusion region found in Bok-S did not differsubstantially from that of the original BH1 domain in Bok-L.Furthermore, the 5 and 6 regions predicted, based on their homology tosimilar regions in Bax and Bak, were unaltered in the truncated Bok-S.These regions have been postulated to be important for channel formationin the mitochondria by different Bcl-2 proteins.

Bok gene arrangement and the derivation of alternative splicingvariants. To elucidate the mechanism by which two Bok isoforms weregenerated, the Bok gene and its exon/intron junctions were analyzed.Following the screening of a bacterial artificial chromosome-based mousegenomic DNA library using a mouse Bok cDNA fragment, one genomic clonefor Bok was isolated. The amino acid sequence of the coding region forthe mouse clone was found to be identical to its rat counterpart.Southern blot hybridization of mouse genomic DNA digested with differentrestriction enzymes using cDNA probes corresponding to two differentregions of the Bok gene demonstrated the presence of a single Bok genein the mouse. Further characterization of the genomic clone indicatedthat the entire Bok gene spanned 11 kb and consisted of 5 exons. The BokORF was encoded by sequences in exons II to V whereas exon I containedonly untranslated sequences. Comparison of the ORF of Bok-S with genomicsequences indicated that Bok-S was derived following the splicing out ofexon III.

Bok-S promotes cell death in transfected cells. To study the role ofBok-S in apoptosis regulation, expression vectors containing Bok-S ineither sense or antisense orientation were constructed. Transfection ofCHO cells with either Bok-S or Bok-L, but not the reverse construct,significantly reduced the number of transfected cells, demonstratingthat Bok-S retained its ability to induce apoptosis despite the loss ofthe BH3 sequence. In addition, cell killing induced by either Bok-S orBok-L was antagonized by cotransfection with P35, a baculoviral-derivedcaspase inhibitor.

Bok-S does not heterodimerize with antiapoptotic Bcl-2 proteins. BecauseBok was isolated based on its ability to bind Mcl-1 and the dimerizationbetween pro- and antiapoptotic Bcl-2 proteins has been suggested to beimportant in apoptosis, we analyzed whether Bok-S that maintained itscell killing ability could still dimerize with antiapoptotic Bcl-2proteins. In the two-hybrid assay, interactions between Bok-S anddifferent Bcl-2 family members were tested. Bok-S did not interact withany Bcl-2 proteins tested whereas Bok-L interacted strongly with Mcl-1,Bfl-1 and BHRF-1, as previously reported. To further confirm findings inyeasts, a direct protein-protein interaction assay was performed usingin vitro translated recombinant Bok variants and the antiapoptoticprotein BHRF-1 that exhibited strongest interaction with Bok-L in yeast.BHRF-1 interacted strongly with Bok-L in vitro but showed negligibleinteraction with Bok-S. Thus, heterodimerization of Bok-S withantiapoptotic Bcl-2 proteins is probably not needed for apoptosisinduction.

BH3 mutants of Bok defective in heterodimerization still retainproapoptotic activity. Because Bok-S lacking a BH3 domain still retainedits cell killing potential, we hypothesized that the BH3 domain might bedispensable for the proapoptotic activity of Bok-L. Bok-L cDNAs withalanine or glycine substitutions in the BH3 domain were constructed andthe ability of these mutants to promote apoptosis was studied. Themutants included alanine substitutions at the highly conserved glycine75 or glycine 75 plus flanking aspartic acid 76 and glutamic acid 77(BokADE: G 75 A and BokAAA: 75 AAA 77). In addition, a glycinesubstitution was made for leucine 71 to leucine 74 (BokGGGG: 71 LLRL 74to 71 GGGG 74). Transfection of these Bok-L mutants reduced the numberof viable CHO cells as compared to the group with cells transfected withthe pcDNA3 vector without an insert. In contrast, constructs with mutantcDNAs in reverse orientation had no effect on cell survival. These datasuggested that the BH3 domain in Bok-L is dispensable for apoptosisinduction. We further tested the ability of these BH3 domain mutants ofBok-L to dimerize with antiapoptotic Bcl-2 proteins in the yeasttwo-hybrid assay. Substitution of residues in the BH3 domain of Bok-Labolished its interaction with Mcl-1 or Bfl-1. In addition, the abilityof Bok-L to interact with BHRF-1 was also abated by glycine substitutionat residues 71-74 of Bok-L. Similar to findings using the two-hybridassay, in vitro translated BokGGGG mutant also lost its ability tointeract with Bok-L effectively in the direct protein-proteininteraction test. These data suggested that the cell killing ability ofthese BH3 mutants is not correlated to their ability to dimerize withantiapoptotic Bcl-2 proteins.

Mutants of Bax and Bak with deletion of their BH3 domain resemblingBok-S also retain proapoptotic activity. Because Bok-L is similar instructure to two other proapoptotic proteins Bax and Bak, deletionmutants with truncation of the BH3-BH1 region similar to that found inBok-S were constructed for these proteins and named as Bax-S and Bak-S(FIG. 3). Full-length Bak and Bax, like Bok-L and Bok-S, effectivelyreduced cell viability in the CHO cell transfection assay. Of interest,overexpression of Bax-S or Bak-S also significantly reduced theviability of transfected cells, suggesting that the BH3-BH1 regionsdeleted in these two proapoptotic proteins are not essential forapoptosis induction.

Discussion

A naturally occurring variant of Bok with proapoptotic activity butexhibiting negligible dimerization with antiapoptotic Bcl-2 members isidentified. Bok-S with a 43-amino acid deletion between the BH3 and BH1domains was likely the result of alternative mRNA splicing, leading tothe skipping of exon three during post-transcriptional modification.Analysis of Bok variants and Bok mutants with alterations in the BH3domain indicated that the BH3 domain of Bok-L is critical forheterodimerization but dispensable for apoptosis induction. Likewise,similar deletions between BH3 and BH1 domains of the homologousproapoptotic proteins Bax and Bak also retained cell killing ability.Thus, Bok-L could promote apoptosis independent of heterodimerizationand Bok-S represents a novel proapoptotic Bcl-2 member capable ofinducing cell death without binding to or interference by antiapoptoticBcl-2 partners. This functional Bok variant with retention of the regionspanning BH1 and BH2 domains and the TM sequence provides a unique modelfor further studies of apoptosis mechanisms regulated by Bcl-2 familyproteins.

The bifunctional antiapoptotic Bcl-2 proteins play a pivotal role in thedecision step of apoptosis. These proteins, represented by Bcl-xL,maintain a channel structure important in the control of mitochondrialmembrane potential and volume homeostasis. Regulation of these channelscontrols the release of cytochrome C essential for the activation ofApaf-1 and caspases important for apoptosis execution. The antiapoptoticBcl-2 proteins also function as docking proteins for proapoptotic Bcl-2members. Because several mutants of Bcl-2 and Bcl-xL simultaneously lostantiapoptotic activity and the ability to bind proapoptotic Bcl-2proteins, it is believed that dimerization of Bcl-2. protein pairsmediated by the consensus BH domains is important in apoptosisregulation. Crystallographic studies and computer modeling showed thatthe conserved BH1, BH2 and BH3 domains of Bcl-xL and related proteinsconstitute an elongated hydrophobic cleft capable of interaction withthe amphipathic helix formed by BH3 domains of proapoptotic partners.Upon heterodimerization, anti- and proapoptotic Bcl-2 partnersantagonize the actions of the other. It is likely that one of themechanisms by which Bok-L exerts its proapoptotic action is throughdimerization with antiapoptotic partners.

Mammalian proapoptotic Bcl-2 proteins can be divided into two subgroupsbased on domain arrangement. Together with Bax and Bak, Bok-L belongs tothe first subgroup showing the conserved BH1, BH2, BH3 and TM domains.In contrast, members of the second subgroup (BAD, BID, Hrk/DP5, Bik/Nbkand Bim) possess only the BH3 domain, with or without the TM region.Earlier studies suggested that proapoptotic proteins function byantagonizing the action of antiapoptotic proteins mediated by BH3domains. Mutations in the BH3 domain of proapoptotic proteins abolishedtheir dimerization with antiapoptotic partners and cell killingactivity. In addition, polypeptides containing minimal BH3 domainsequences bind antiapoptotic proteins and induce apoptosis intransfected cells or cell-free systems. More recent studies, however,demonstrated that Bax, like Bcl-xL and Bcl-2, also shows intrinsic ionchannel activity in the artificial membrane. In addition, mutations inthe BH1, 2 or 3 domains of Bax do not affect its ability to, promoteapoptosis. Likewise, Bak mutants accelerate chemotherapy-inducedapoptosis independent of its heterodimerization property. These datasuggest that the first subgroup of proapoptotic proteins, including Bax,Bak and Bok, could induce apoptosis through channel formation inaddition to their role as ligands for antiapoptotic Bcl-2 proteins.Because the second BH3-only subgroup members lack the region spanningBH1 and BH2 domains important for pore formation and mainly reside inthe cytoplasm, they are believed to serve as ligands or facilitators ofthe pore forming Bcl-2 proteins.

Our findings that substitution of conserved residues in the BH3 domainof Bok-L abates its ability to dimerize with antiapoptotic proteins arein accord with studies on the BH3 domain of its proapoptotic homologues.Similarly, truncation of the conserved BH3 domain in thenaturally-occurring Bok-S variant also disrupted heterodimerization butretained cell killing ability, indicating the BH3 domain is dispensablefor apoptosis induction. Thus, Bok-S represents a new form ofproapoptotic protein consisting of only minimal functional modules andmanifesting proapoptotic action without direct interactions withantiapoptotic proteins. As shown in the above data, truncation of theregion between BH3 and BH1 from Bok-L does not affect the homologous α5and α6 regions proposed to be important for channel formation in Bax. Inaddition, the hydropathicity property between the 5′-end of the BH1region and the C-terminal TM domain is not altered by the truncationfound in Bok-S. It is likely that the BH3/1, BH2 and TM domains found inBok-S comprise a module sufficient for mediating apoptosis through aheterodimerization-independent pathway. Future studies on thechannel-forming property of the naturally-occurring Bok-S and otherchannel-forming Bcl-2 proteins are important for understanding themechanisms of apoptosis. The channel-forming hypothesis is furthersupported by the finding that Bax-S and Bak-S with truncation at theBH3-BH1 regions homologous to that of Bok-S also retain proapoptoticactivity. Recent studies also indicated that, during apoptosis,activated caspases cleave the N-terminal BH4 domain of antiapoptoticproteins Bcl-2 and Bcl-xL to yield truncated molecules resembling theproapoptotic Bax, Bak or Bok in terms of the BH domain arrangement. Ofinterest, deletion of the BH4 domain from these antiapoptotic proteinsconfers proapoptotic activity and mitochondrial release of cytochrome C,presumably mediated through the C-terminal channel-forming region.

Like Bok, splicing variants have been reported for Bcl-2, Bcl-x and Baxgenes. The Bcl-xL gene encodes three different variants, each with adistinct function; the long form of Bcl-x (L) exhibits antiapoptoticactivity whereas Bcl-x-short and Bcl-x- are proapoptotic. Also, Bcl-2variants lacking the TM domain show decreased antiapoptotic activity.The proapoptotic Bax gene also encodes a number of splicing variantswith unknown function.

At least three mechanisms could be proposed for the action ofproapoptotic Bcl-2 proteins: 1) The subgroup of proapoptotic proteinswith only the BH3 domain (e.g. the soluble BAD protein) heterodimerizeswith membrane-bound antiapoptotic proteins to regulate apoptosis; 2) Thesubgroup of membrane-bound proapoptotic proteins with BH1, BH2, BH3 andTM domains, represented by Bok-L, heterodimerizes with antiapoptoticproteins (Mcl-1/Bfl-1) or functions as mitochondrial channels toregulate apoptosis; and 3) The unique Bok-S variant does not dimerizewith antiapoptotic proteins but probably forms mitochondrial channels toregulate apoptosis. Because Bok-S does not interact with antiapoptoticproteins, apoptosis mediated through Bok-S may be important insituations when unwanted cells need to be eliminated quickly despite thepresence of antiapoptotic proteins in the same cell. In the ovary anduterus known to express high levels of Bok transcripts, Bok-S expressioncould provide a short circuit to promote cell demise inhormone-dependent cell populations that express abundant antiapoptoticproteins (such as Mcl -1) but have to be removed swiftly due to cycliccell turnover during reproductive cycles. The search for novel deathpromoters that interact specifically with proapoptotic Bcl-2 proteinscould also be, simplified based on the lack of interaction between Bok-Sand other Bcl-2 proteins. Further characterization of this uniqueproapoptotic protein would allow a better understanding of intracellularmechanism of apoptosis, particularly for hormone-regulated cell death.

18 1 642 DNA r. rattus 1 atggaggtgc tgcggcgctc ttctgtcttc gctgcggagatcatggacgc ctttgatcgc 60 tcgcccacag acaaggagct ggtggcccag gctaaagcactaggccggga gtacgtgcac 120 gcgcggcttt tgcgcgccgg cctctcctgg agcgctccagagcgtgcctc gcctgcccct 180 ggaggacgcc tggcagaggt gtgcaccgtg ctgctgcgcttgggagatga gctggagcag 240 atccgtccca gcgtatatcg gaatgtggcc cggcagctgcacatccccct gcagtctgag 300 cctgtggtga ctgatgcctt cctcgctgtg gccggccacatcttctcagc aggtatcaca 360 tggggcaagg tagtgtccct gtactcggtg gctgcgggactagcggtgga ctgcgtccgg 420 caagctcagc cagccatggt tcatgccctg gttgactgcctgggggaatt tgtacgcaag 480 accctggcca cctggcttcg gaggcgtggt ggatggacggacgtcctcaa gtgtgtggtc 540 agcacagacc ctggcttccg ctcccactgg ctcgtggccacactctgcag ctttggccgc 600 ttcctgaagg ctgcattctt cctcctgttg ccagagagat ga642 2 213 PRT r. rattus 2 Met Glu Val Leu Arg Arg Ser Ser Val Phe AlaAla Glu Ile Met Asp 1 5 10 15 Ala Phe Asp Arg Ser Pro Thr Asp Lys GluLeu Val Ala Gln Ala Lys 20 25 30 Ala Leu Gly Arg Glu Tyr Val His Ala ArgLeu Leu Arg Ala Gly Leu 35 40 45 Ser Trp Ser Ala Pro Glu Arg Ala Ser ProAla Pro Gly Gly Arg Leu 50 55 60 Ala Glu Val Cys Thr Val Leu Leu Arg LeuGly Asp Glu Leu Glu Gln 65 70 75 80 Ile Arg Pro Ser Val Tyr Arg Asn ValAla Arg Gln Leu His Ile Pro 85 90 95 Leu Gln Ser Glu Pro Val Val Thr AspAla Phe Leu Ala Val Ala Gly 100 105 110 His Ile Phe Ser Ala Gly Ile ThrTrp Gly Lys Val Val Ser Leu Tyr 115 120 125 Ser Val Ala Ala Gly Leu AlaVal Asp Cys Val Arg Gln Ala Gln Pro 130 135 140 Ala Met Val His Ala LeuVal Asp Cys Leu Gly Glu Phe Val Arg Lys 145 150 155 160 Thr Leu Ala ThrTrp Leu Arg Arg Arg Gly Gly Trp Thr Asp Val Leu 165 170 175 Lys Cys ValVal Ser Thr Asp Pro Gly Phe Arg Ser His Trp Leu Val 180 185 190 Ala ThrLeu Cys Ser Phe Gly Arg Phe Leu Lys Ala Ala Phe Phe Leu 195 200 205 LeuLeu Pro Glu Arg 210 3 513 DNA r.rattus CDS (1)...(513) 3 atg gag gtg ctgcgg cgc tct tct gtc ttc gct gcg gag atc atg gac 48 Met Glu Val Leu ArgArg Ser Ser Val Phe Ala Ala Glu Ile Met Asp 1 5 10 15 gcc ttt gat cgctcg ccc aca gac aag gag ctg gtg gcc cag gct aaa 96 Ala Phe Asp Arg SerPro Thr Asp Lys Glu Leu Val Ala Gln Ala Lys 20 25 30 gca cta ggc cgg gagtac gtg cac gcg cgg ctt ttg cgc gcc ggc ctc 144 Ala Leu Gly Arg Glu TyrVal His Ala Arg Leu Leu Arg Ala Gly Leu 35 40 45 tcc tgg agc gct cca gagcgt gcc tcg cct gcc cct gga gga cgc ctg 192 Ser Trp Ser Ala Pro Glu ArgAla Ser Pro Ala Pro Gly Gly Arg Leu 50 55 60 gca gag gtg tgc acc gtg ctgctg cgc ttg gga atc aca tgg ggc aag 240 Ala Glu Val Cys Thr Val Leu LeuArg Leu Gly Ile Thr Trp Gly Lys 65 70 75 80 gta gtg tcc ctg tac tcg gtggct gcg gga cta gcg gtg gac tgc gtc 288 Val Val Ser Leu Tyr Ser Val AlaAla Gly Leu Ala Val Asp Cys Val 85 90 95 cgg caa gct cag cca gcc atg gttcat gcc ctg gtt gac tgc ctg ggg 336 Arg Gln Ala Gln Pro Ala Met Val HisAla Leu Val Asp Cys Leu Gly 100 105 110 gaa ttt gta cgc aag acc ctg gccacc tgg ctt cgg agg cgt ggt gga 384 Glu Phe Val Arg Lys Thr Leu Ala ThrTrp Leu Arg Arg Arg Gly Gly 115 120 125 tgg acg gac gtc ctc aag tgt gtggtc agc aca gac cct ggc ttc cgc 432 Trp Thr Asp Val Leu Lys Cys Val ValSer Thr Asp Pro Gly Phe Arg 130 135 140 tcc cac tgg ctc gtg gcc aca ctctgc agc ttt ggc cgc ttc ctg aag 480 Ser His Trp Leu Val Ala Thr Leu CysSer Phe Gly Arg Phe Leu Lys 145 150 155 160 gct gca ttc ttc ctc ctg ttgcca gag aga tga 513 Ala Ala Phe Phe Leu Leu Leu Pro Glu Arg * 165 170 4170 PRT r.rattus 4 Met Glu Val Leu Arg Arg Ser Ser Val Phe Ala Ala GluIle Met Asp 1 5 10 15 Ala Phe Asp Arg Ser Pro Thr Asp Lys Glu Leu ValAla Gln Ala Lys 20 25 30 Ala Leu Gly Arg Glu Tyr Val His Ala Arg Leu LeuArg Ala Gly Leu 35 40 45 Ser Trp Ser Ala Pro Glu Arg Ala Ser Pro Ala ProGly Gly Arg Leu 50 55 60 Ala Glu Val Cys Thr Val Leu Leu Arg Leu Gly IleThr Trp Gly Lys 65 70 75 80 Val Val Ser Leu Tyr Ser Val Ala Ala Gly LeuAla Val Asp Cys Val 85 90 95 Arg Gln Ala Gln Pro Ala Met Val His Ala LeuVal Asp Cys Leu Gly 100 105 110 Glu Phe Val Arg Lys Thr Leu Ala Thr TrpLeu Arg Arg Arg Gly Gly 115 120 125 Trp Thr Asp Val Leu Lys Cys Val ValSer Thr Asp Pro Gly Phe Arg 130 135 140 Ser His Trp Leu Val Ala Thr LeuCys Ser Phe Gly Arg Phe Leu Lys 145 150 155 160 Ala Ala Phe Phe Leu LeuLeu Pro Glu Arg 165 170 5 642 DNA H.sapiens CDS (1)...(642) 5 atg gaggtg ctg cgg cgc tct tcg gtc ttc gct gcg gag atc atg gac 48 Met Glu ValLeu Arg Arg Ser Ser Val Phe Ala Ala Glu Ile Met Asp 1 5 10 15 gcc tttgat cgc tgg ccc aca gac aag gag ctg gtg gcc cag gct aaa 96 Ala Phe AspArg Trp Pro Thr Asp Lys Glu Leu Val Ala Gln Ala Lys 20 25 30 gca cta ggccgg gag tac gtg cac gcg cgg ctt ttg cgc gcc ggc ctc 144 Ala Leu Gly ArgGlu Tyr Val His Ala Arg Leu Leu Arg Ala Gly Leu 35 40 45 tcc tgg agc gctcca gag cgt gcc tcg cct gcc cct gga gga cgc ctg 192 Ser Trp Ser Ala ProGlu Arg Ala Ser Pro Ala Pro Gly Gly Arg Leu 50 55 60 gca gag gtg tgc accgtg ctg ctg cgc ttg gga gat gag ctg gag cag 240 Ala Glu Val Cys Thr ValLeu Leu Arg Leu Gly Asp Glu Leu Glu Gln 65 70 75 80 atc cgt ccc agc gtatat cgg aat gtg gcc cgg cag ctg cac atc cct 288 Ile Arg Pro Ser Val TyrArg Asn Val Ala Arg Gln Leu His Ile Pro 85 90 95 ctg cag tct gag cct gtggtg act gat gcc ttc ctc gct gtg gcc ggc 336 Leu Gln Ser Glu Pro Val ValThr Asp Ala Phe Leu Ala Val Ala Gly 100 105 110 cac atc ttc tca gca ggtatc aca tgg ggc aag gta gtg tcc ctg tac 384 His Ile Phe Ser Ala Gly IleThr Trp Gly Lys Val Val Ser Leu Tyr 115 120 125 tcg gcg gct gcg gga ctagcg gtg gac tgc gtc cgg caa gct cag cca 432 Ser Ala Ala Ala Gly Leu AlaVal Asp Cys Val Arg Gln Ala Gln Pro 130 135 140 gcc atg gtt cat gcc ctggtt gac tgc ctg ggg gaa ttt gta cgc aag 480 Ala Met Val His Ala Leu ValAsp Cys Leu Gly Glu Phe Val Arg Lys 145 150 155 160 acc ttg gct acc tggctt cgg agg cgt ggt gga tgg acg gac gtc ctc 528 Thr Leu Ala Thr Trp LeuArg Arg Arg Gly Gly Trp Thr Asp Val Leu 165 170 175 aag tgt gtg gtc agcaca aaa cct ggc ttc cgc tcc cac tgg ctc gtg 576 Lys Cys Val Val Ser ThrLys Pro Gly Phe Arg Ser His Trp Leu Val 180 185 190 gcc aca ctc tgc agcttt ggc cgc ttc ctg aag gct gca ttc ttc ctc 624 Ala Thr Leu Cys Ser PheGly Arg Phe Leu Lys Ala Ala Phe Phe Leu 195 200 205 ctg ttg cca gag agatga 642 Leu Leu Pro Glu Arg * 210 6 213 PRT H.sapiens 6 Met Glu Val LeuArg Arg Ser Ser Val Phe Ala Ala Glu Ile Met Asp 1 5 10 15 Ala Phe AspArg Trp Pro Thr Asp Lys Glu Leu Val Ala Gln Ala Lys 20 25 30 Ala Leu GlyArg Glu Tyr Val His Ala Arg Leu Leu Arg Ala Gly Leu 35 40 45 Ser Trp SerAla Pro Glu Arg Ala Ser Pro Ala Pro Gly Gly Arg Leu 50 55 60 Ala Glu ValCys Thr Val Leu Leu Arg Leu Gly Asp Glu Leu Glu Gln 65 70 75 80 Ile ArgPro Ser Val Tyr Arg Asn Val Ala Arg Gln Leu His Ile Pro 85 90 95 Leu GlnSer Glu Pro Val Val Thr Asp Ala Phe Leu Ala Val Ala Gly 100 105 110 HisIle Phe Ser Ala Gly Ile Thr Trp Gly Lys Val Val Ser Leu Tyr 115 120 125Ser Ala Ala Ala Gly Leu Ala Val Asp Cys Val Arg Gln Ala Gln Pro 130 135140 Ala Met Val His Ala Leu Val Asp Cys Leu Gly Glu Phe Val Arg Lys 145150 155 160 Thr Leu Ala Thr Trp Leu Arg Arg Arg Gly Gly Trp Thr Asp ValLeu 165 170 175 Lys Cys Val Val Ser Thr Lys Pro Gly Phe Arg Ser His TrpLeu Val 180 185 190 Ala Thr Leu Cys Ser Phe Gly Arg Phe Leu Lys Ala AlaPhe Phe Leu 195 200 205 Leu Leu Pro Glu Arg 210 7 513 DNA H. sapiens CDS(1)...(513) 7 atg gag gtg ctg cgg cgc tct tcg gtc ttc gct gcg gag atcatg gac 48 Met Glu Val Leu Arg Arg Ser Ser Val Phe Ala Ala Glu Ile MetAsp 1 5 10 15 gcc ttt gat cgc tgg ccc aca gac aag gag ctg gtg gcc caggct aaa 96 Ala Phe Asp Arg Trp Pro Thr Asp Lys Glu Leu Val Ala Gln AlaLys 20 25 30 gca cta ggc cgg gag tac gtg cac gcg cgg ctt ttg cgc gcc ggcctc 144 Ala Leu Gly Arg Glu Tyr Val His Ala Arg Leu Leu Arg Ala Gly Leu35 40 45 tcc tgg agc gct cca gag cgt gcc tcg cct gcc cct gga gga cgc ctg192 Ser Trp Ser Ala Pro Glu Arg Ala Ser Pro Ala Pro Gly Gly Arg Leu 5055 60 gca gag gtg tgc acc gtg ctg ctg cgc ttg gga atc aca tgg ggc aag240 Ala Glu Val Cys Thr Val Leu Leu Arg Leu Gly Ile Thr Trp Gly Lys 6570 75 80 gta gtg tcc ctg tac tcg gcg gct gcg gga cta gcg gtg gac tgc gtc288 Val Val Ser Leu Tyr Ser Ala Ala Ala Gly Leu Ala Val Asp Cys Val 8590 95 cgg caa gct cag cca gcc atg gtt cat gcc ctg gtt gac tgc ctg ggg336 Arg Gln Ala Gln Pro Ala Met Val His Ala Leu Val Asp Cys Leu Gly 100105 110 gaa ttt gta cgc aag acc ttg gct acc tgg ctt cgg agg cgt ggt gga384 Glu Phe Val Arg Lys Thr Leu Ala Thr Trp Leu Arg Arg Arg Gly Gly 115120 125 tgg acg gac gtc ctc aag tgt gtg gtc agc aca aaa cct ggc ttc cgc432 Trp Thr Asp Val Leu Lys Cys Val Val Ser Thr Lys Pro Gly Phe Arg 130135 140 tcc cac tgg ctc gtg gcc aca ctc tgc agc ttt ggc cgc ttc ctg aag480 Ser His Trp Leu Val Ala Thr Leu Cys Ser Phe Gly Arg Phe Leu Lys 145150 155 160 gct gca ttc ttc ctc ctg ttg cca gag aga tga 513 Ala Ala PhePhe Leu Leu Leu Pro Glu Arg * 165 170 8 170 PRT H. sapiens 8 Met Glu ValLeu Arg Arg Ser Ser Val Phe Ala Ala Glu Ile Met Asp 1 5 10 15 Ala PheAsp Arg Trp Pro Thr Asp Lys Glu Leu Val Ala Gln Ala Lys 20 25 30 Ala LeuGly Arg Glu Tyr Val His Ala Arg Leu Leu Arg Ala Gly Leu 35 40 45 Ser TrpSer Ala Pro Glu Arg Ala Ser Pro Ala Pro Gly Gly Arg Leu 50 55 60 Ala GluVal Cys Thr Val Leu Leu Arg Leu Gly Ile Thr Trp Gly Lys 65 70 75 80 ValVal Ser Leu Tyr Ser Ala Ala Ala Gly Leu Ala Val Asp Cys Val 85 90 95 ArgGln Ala Gln Pro Ala Met Val His Ala Leu Val Asp Cys Leu Gly 100 105 110Glu Phe Val Arg Lys Thr Leu Ala Thr Trp Leu Arg Arg Arg Gly Gly 115 120125 Trp Thr Asp Val Leu Lys Cys Val Val Ser Thr Lys Pro Gly Phe Arg 130135 140 Ser His Trp Leu Val Ala Thr Leu Cys Ser Phe Gly Arg Phe Leu Lys145 150 155 160 Ala Ala Phe Phe Leu Leu Leu Pro Glu Arg 165 170 9 12 PRTR. rattus 9 Leu Leu Arg Leu Gly Asp Glu Leu Glu Gln Ile Arg 1 5 10 10 12PRT H. sapiens 10 Leu Leu Arg Leu Gly Ile Thr Trp Gly Lys Val Val 1 5 1011 13 PRT Artificial Sequence Consensus motif 11 Leu Arg Arg Ala Gly AspGlu Phe Glu Arg Tyr Arg Arg 1 5 10 12 4 PRT H. sapiens 12 Thr Trp GlyLys 1 13 4 PRT H. sapiens 13 Asn Trp Gly Arg 1 14 3 PRT H. sapiens 14Gly Asp Glu 1 15 24 PRT H. sapiens 15 Leu Arg Arg Ile Gly Asp Glu LeuAsp Ser Asn Ala Asp Gly Asn Phe 1 5 10 15 Asn Trp Gly Arg Val Val AlaLeu 20 16 24 PRT H. sapiens 16 Leu Ala Ile Ile Gly Asp Asp Ile Asn ArgArg Phe Glu Ser Gly Ile 1 5 10 15 Asn Trp Gly Arg Val Val Ala Leu 20 1713 PRT H. sapiens 17 Leu Arg Arg Ile Gly Asn Phe Asn Trp Gly Arg Val Val1 5 10 18 12 PRT H. sapiens 18 Leu Ala Ile Ile Gly Ile Asn Trp Gly ArgVal Val 1 5 10

What is claimed is:
 1. A purified polypeptide composition comprising atleast 50 weight % of the composition present as a Bok protein as setforth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or SEQ ID NO:8.
 2. Apurified polypeptide comprising a fragment of at least 16 amino acids ofa sequence within any one of SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6; orSEQ ID NO:8.
 3. The purified polypeptide fragment of claim 2, whereinsaid fragment is immunogenic.
 4. The purified polypeptide fragment ofclaim 2, wherein said fragment comprises a BH domain.
 5. The polypeptideaccording to claim 2, wherein said fragment is a pro-apoptotic variantprotein.
 6. The purified polypeptide fragment of claim 4, wherein saidfragment comprises a BH3 domain.
 7. The polypeptide compositionaccording to claim 5, wherein said variant is a BH3^(i) variant.